Heterotropic cooperativity within and between protomers of an oligomeric M(2) muscarinic receptor.

At least four allosteric sites have been found to mediate the dose-dependent effects of gallamine on the binding of [(3)H]quinuclidinylbenzilate (QNB) and N-[(3)H]methylscopolamine (NMS) to M(2) muscarinic receptors in membranes and solubilized preparations from porcine atria, CHO cells, and Sf9 cells. The rate of dissociation of [(3)H]QNB was affected in a bell-shaped manner with at least one Hill coefficient (n(H)) greater than 1, indicating that at least three allosteric sites are involved. The level of binding of [(3)H]QNB was decreased in a biphasic manner, revealing at least two allosteric sites; binding of [(3)H]NMS was affected in a triphasic, serpentine manner, revealing at least three sites, and values of n(H) >1 pointed to at least four sites. Several lines of evidence indicate that all effects of gallamine were allosteric in nature and could be observed at equilibrium. The rates of equilibration and dissociation suggest that the receptor was predominately oligomeric, and the heterogeneity revealed by gallamine can be attributed to differences in its affinity for the constituent protomers of a tetramer. Those differences appear to arise from inter- and intramolecular cooperativity between gallamine and the radioligand.

Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist.

The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.

Intracellular distribution of functional M(1) -muscarinic acetylcholine receptors in N1E-115 neuroblastoma cells.

Signaling by muscarinic agonists is thought to result from the activation of cell surface acetylcholine receptors (mAChRs) that transmit extracellular signals to intracellular systems. In N1E-115 neuroblastoma cells, we detected both plasma membrane and intracellular M(1) -mAChRs using both biochemical and pharmacological methods. In intact cells, both plasma membrane and intracellular M(1) -mAChRs were detected by the hydrophobic ligand probe, 1-quinuclidinyl-[phenyl-4-(3) H]-benzilate ([(3) H]-QNB) whereas the hydrophilic probe, 1-[N-methyl-(3) H] scopolamine ([(3) H]-NMS), detected only cell surface receptors. These probes detected comparable numbers of receptors in isolated membrane preparations. Immunohistochemical studies with M(1) -mAChR antibody also detected both cell-surface and intracellular M(1) -mAChRs. Carbachol-stimulated phosphatidylinositol hydrolysis and Ca(2+) mobilization were completely inhibited by a cell-impermeable M(1) antagonist, muscarinic toxin -7 and the G(q/11) inhibitor YM-254890. However, carbachol-stimulated extracellular-regulated kinase 1/2 activation was unaffected by muscarinic toxin-7, but was blocked by the cell-permeable antagonist, pirenzepine. extracellular regulated kinase 1/2 phosphorylation was resistant to blockade of G(q/11) (YM-254890) and protein kinase C (bisindolylmaleimide I). Our data suggest that the geographically distinct M(1) -mAChRs (cell surface versus intracellular) can signal via unique signaling pathways that are differentially sensitive to cell-impermeable versus cell-permeable antagonists. Our data are of potential physiological relevance to signaling that affects both cognitive and neurodegenerative processes.

Heterooligomers of the muscarinic receptor and G proteins purified from porcine atria.

Muscarinic receptor extracted from porcine atria in digitonin-cholate copurified with Galpha(o), Galpha(i1-3), and caveolins. The presence of complexes was confirmed by coimmunoprecipitation of the receptor, alpha-subunits, and caveolins in various combinations. Homooligomers of alpha(i2) were detected on Western blots, and heterooligomers of alpha(i2) and alpha(o) were identified by coimmunoprecipitation; thus, a complex may contain at least two alpha-subunits. Other combinations of alpha-subunit were not detected. The ratio of total alpha-subunit to receptor was near 1, as measured by [(35)S]GTPgammaS and the antagonist [(3)H]quinuclidinylbenzilate, and the binding of [(35)S]GTPgammaS was manifestly biphasic. The ratio of alpha(o) to alpha(i1,2) also was near 1, as determined from the intensity of Western blots. Cardiac muscarinic receptors therefore can be purified as a mixture of complexes that contain caveolins and oligomers of alpha-subunit, some of which are heteromeric. Each complex would appear to contain equal numbers of alpha-subunit and the receptor.

New insights into the function of M4 muscarinic acetylcholine receptors gained using a novel allosteric modulator and a DREADD (designer receptor exclusively activated by a designer drug).

The M4 muscarinic acetylcholine (ACh) receptor (mAChR) is a potential therapeutic target but characterized by a lack of subtype-selective ligands. We recently generated "designer receptors exclusively activated by a designer drug" (DREADDs), which contained mutations of two conserved orthosteric-site residues (Y113C/A203G in the M4 mAChR) that caused a loss of ACh activity but a gain in responsiveness to clozapine-N-oxide (CNO). The current study characterized the interactions of the wild type and the M4 DREADD with a range of agonists, antagonists, and the recently discovered M4 mAChR allosteric potentiator, 3-amino-5-chloro-6-methoxy-4-methyl-thieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298). LY2033298 displayed positive binding cooperativity with ACh, neutral cooperativity with the antagonist, [3H]quinuclidinyl benzilate, and agonism for activation of phosphorylated extracellular signal-regulated kinase (ERK) 1/2 at the wild-type M4 mAChR. LY2033298's cooperativity with clozapine or CNO was weakly positive with respect to binding but profoundly negative with respect to LY2033298 signaling. Although the DREADD mutations increased the binding and function of clozapine-like compounds, all other agonists lost the ability to activate the mutant; for the orthosteric agonists ACh and pilocarpine, this was due partly to a reduced affinity, whereas the affinity of LY2033298 or the atypical agonist 4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride was unaltered. The interaction between LY2033298 and clozapine-like compounds reverted to neutral cooperativity on the DREADD, whereas LY2033298 caused a striking functional rescue of ACh potency and efficacy at the DREADD. These results provide conclusive evidence for the retention of a functional allosteric site on the M4 DREADD and highlight a role for residues Tyr113 and Ala203 in the transmission of cooperativity.

[Propofol-induced myocardial depression: possible role of atrial muscarinic cholinergic receptors]. / Depresión de la función mioc irdica inducida por propofol: posible participación del receptor colinérgico muscarínico auricular.

OBJECTIVE: To investigate the possible role of muscarinic cholinergic receptors (MCRs) in the depression of myocardial function induced by propofol, an intravenous anesthetic chemically unrelated to other drugs. Although adverse effects are rare, bradycardia has been reported and this can lead to cardiac arrest in some patients. The mechanism behind this effect is still unknown but a possible role for MCRs has been suggested. MATERIAL AND METHODS: The interaction of propofol with human atrial MCRs was determined by means of inhibition tests using [3H] quinuclidinyl benzilate ([3H] QNB). RESULTS: The displacement of [3H] QNB binding to human atrial MCRs by propofol was concentration dependent but the observed effect was not consistent with a model of simple competition between propofol and [3H] QNB. CONCLUSION: Propofol appears to have the ability to modify the activity of human atrial MCRs and this effect may be related to its ability to induce bradycardia.

Stress and re-stress increases conditioned taste aversion learning in rats: possible frontal cortical and hippocampal muscarinic receptor involvement.

Symptoms of posttraumatic stress disorder are often precipitated by sensory cues in the form of visual, auditory, olfactory and gustatory "flashbacks" resulting in enhanced fear-memory consolidation and the characteristic symptoms of re-experiencing, avoidance and hyper-arousal. Single prolonged stress with and without re-stress have been used to explore the neurobiology of this disorder, particularly with respect to contextual conditioning and spatial memory impairment. However, less work has been done regarding associative sensory-related memories linked to aversive events. Although growing evidence supports a role for cholinergic pathways in stress, this has not been studied in the above animal models. We studied the effects of single prolonged stress with and without re-stress on conditioned taste aversion learning in rats, together with differential analysis of frontal cortical and hippocampal [3H]-quinuclidinyl benzylate ([3H]-QNB) muscarinic receptor binding. Single prolonged stress with and without re-stress both enhanced associative sensory aversion learning 7 days after stressor-taste pairing, although re-stress did not strengthen this response. Increased cortical and hippocampal muscarinic receptor density (Bmax) was found 7 days after single prolonged stress with re-stress, although receptor affinity remained unaltered. Frontal cortical and hippocampal muscarinic receptor changes may thus underlie conditioned taste aversion learning in rats exposed to stress and re-stress. These data suggest that it may be useful to study the role of cholinergic pathways in mediating associative memory in psychiatric disorders such as posttraumatic stress disorder.

The cholinergic nervous system plays an important role in rat postoperative intestinal adhesion.

BACKGROUND: Postoperative adhesions can cause serious complications after abdominal surgery. This study demonstrates the role of the cholinergic nervous system in the development of postoperative intestinal adhesion. METHODS: Postoperative intestinal adhesion was induced by sprinkling talc on the small intestines of rats, and the adhesion rate, histology, and gastrointestinal transit were evaluated. To investigate the involvement of the cholinergic nervous system in postoperative intestinal adhesion, we evaluated choline acetyltransferase (ChAT) activity, muscarinic receptor density, and the preventive effect of a muscarine receptor agonist, bethanechol, on talc-induced intestinal adhesion in rats. RESULTS: Histologic examination revealed inflammation in the intestinal adhesion regions, but no damage was seen in sham-operated rats. The rate of adhesion formation had significantly increased 3-7 days after surgery. The gastrointestinal transit was decreased by about 30% in the talc-induced intestinal adhesion rats. ChAT activity decreased by about 50% in adhesion regions. In contrast, the density of muscarinic receptors was higher in rats with talc-induced intestinal adhesions. Furthermore, bethanechol significantly prevented 30%-41% of adhesion formation in rats with talc-induced intestinal adhesions. This action was inhibited by subcutaneous injection of atropine. CONCLUSIONS: Postoperative intestinal adhesion affected the cholinergic nervous system as demonstrated by decreased ChAT activity and increased density of muscarinic receptors. These alterations of gastrointestinal function might be a cause of adhesion formation.

Comparative in vivo effects of parathion on striatal acetylcholine accumulation in adult and aged rats.

Aged rats are more sensitive to the acute toxicity of the prototype organophosphate insecticide, parathion. We compared the acute effects of parathion on diaphragm and brain regional cholinesterase activity, muscarinic receptor binding and striatal acetylcholine levels in 3- and 18-month-old male Sprague-Dawley rats. Adult and aged rats were surgically implanted with a microdialysis cannula into the right striatum 5-7 days prior to parathion treatment. Rats were given either vehicle (peanut oil, 2 ml/kg) or one of a range of dosages of parathion (adult: 1.8, 3.4, 6.0, 9.0, 18 and 27 mg/kg, s.c.; aged: 1.8, 3.4, 6 and 9 mg/kg, s.c.) and body weight, functional signs of toxicity, and nocturnal motor activity were recorded for seven days. Three and seven days after parathion treatment, microdialysis samples were collected and rats were subsequently sacrificed for biochemical measurements. Higher dosages of parathion led to significant time-dependent reductions in body weight in both age groups. Rats in both age groups treated with lower dosages showed few overt signs of cholinergic toxicity while equitoxic high dosages (adult, 27 mg/kg; aged, 9 mg/kg) elicited marked signs of cholinergic toxicity (involuntary movements and SLUD [i.e., acronym for Salivation, Lacrimation, Urination and Defecation] signs) with peak effects being noted 3-4 days after treatment. Nocturnal activity (ambulation and rearing) was reduced in both age groups following parathion dosing, with more prominent effects in adults and rearing being more consistently affected. Dose- and time-dependent inhibition of cholinesterase activity was noted in both diaphragm and striatum. Total muscarinic receptor ([(3)H]quinuclidinyl benzilate, QNB) binding was significantly lower in aged rats, and both total binding and muscarinic agonist ([(3)H]oxotremorine methiodide] binding was significantly reduced in both age-groups treated with the highest dosages of parathion (adult, 27 mg/kg; aged, 9 mg/kg). In contrast to relatively similar levels of cholinesterase inhibition, striatal extracellular acetylcholine levels were significantly lower (2.2- to 2.9-fold) in aged rats at both 3 and 7 day time-points compared to adult rats treated with equitoxic dosages (i.e., 9 and 27 mg/kg, respectively). No age-related differences in in vitro striatal acetylcholine synthesis or in vivo acetylcholine accumulation following direct infusion of the cholinesterase inhibitor neostigmine (1 microM) were noted. While aged rats are more sensitive than adults to the acute toxicity of parathion, lesser acetylcholine accumulation was noted in the striatum of aged rats exhibiting similar levels of cholinesterase inhibition. These findings suggest that lesser acetylcholine accumulation may be required to elicit cholinergic signs in the aged rat, possibly based on aging-associated changes in muscarinic receptor density.

Muscarinic receptors autoantibodies purified from mammary adenocarcinoma-bearing mice sera stimulate tumor progression.

The ability of tumor cells to stimulate adaptive immunity, particularly by inducing anti-tumor antibodies (Abs), has been extensively reviewed. LM3 is a tumorigenic cell line derived from a murine mammary metastatic adenocarcinoma that spontaneously overexpressed mAchR. Here we investigate the ability of Abs purified from the sera of LM3 tumor-bearing mice, directed against muscarinic acetylcholine receptors (mAchR) to modulate tumor cells' proliferation and angiogenesis. We observed that IgG from early tumor bearers (ETB), 14-day LM3 tumor, and from late tumor bearers (LTB), 28-day LM3 tumor, displaced tritiated quinuclidinyl benzilate binding to LM3 tumor cells, confirming Abs interaction with cholinoceptors, while IgG from normal mice did not modify the antagonist binding to mAchR at any concentration tested. In addition, Abs from ETB and LTB immunoblotted a protein of 70 kDa on murine tumor cells and on heart homogenates that was also recognized by a specific anti-M(2) receptor monoclonal antibody. We also observed that IgG purified from ETB-stimulated LM3 cells' proliferation in a more effective manner than the muscarinic agonist carbachol (CARB) did. IgG from LTB-potentiated LM3 cells induced angiogenesis by increasing the number of blood vessels and VEGF-A production in peritumoral skin "via" mAchR, in an agonist similar manner. All effects were blocked by preincubating cells with the non-selective antagonist atropine. In conclusion, autoAbs purified from LM3 tumor-bearing mice sera exert different pro-tumor actions depending on the stage of tumor development: in ETB, they stimulate tumor cells' proliferation, while in LTB they potentiate tumor neovascularization.

Preparation and biological effects of pure stereoisomeric novel soft anticholinergics.

A series of pure stereoisomeric soft glycopyrrolate analogues 3, 4 and 5 was synthesized using chiral intermediates and by careful separation of the stereoisomers formed during the last quaternization step of the synthesis. The stereochemistry of the products was elucidated using various 1D and 2D NMR techniques. Anticholinergic activity of the new compounds was determined by receptor binding studies and performing tests on isolated organs and by in vivo tests. Receptor binding revealed that in the higher alkyl ester series the (2R, 1'R, 3'R) and the (2R, 1'S, 3'S) isomers were the compounds showing the highest receptor affinity furthermore it demonstrated the confines of the length of the alkyl chain. In vitro isolated organ experiments correlated well with the receptor binding results, and in vivo investigations indicated the soft character of the compounds.

In vivo co-ordinated interactions between inhibitory systems to control glutamate-mediated hippocampal excitability.

We present an overview of the long-term adaptation of hippocampal neurotransmission to cholinergic and GABAergic deafferentation caused by excitotoxic lesion of the medial septum. Two months after septal microinjection of 2.7 nmol alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), a 220% increase of GABA(A) receptor labelling in the hippocampal CA3 and the hilus was shown, and also changes in hippocampal neurotransmission characterised by in vivo microdialysis and HPLC. Basal amino acid and purine extracellular levels were studied in control and lesioned rats. In vivo effects of 100 mm KCl perfusion and adenosine A(1) receptor blockade with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) on their release were also investigated. In lesioned animals GABA, glutamate and glutamine basal levels were decreased and taurine, adenosine and uric acid levels increased. A similar response to KCl infusion occurred in both groups except for GABA and glutamate, which release decreased in lesioned rats. Only in lesioned rats, DPCPX increased GABA basal level and KCl-induced glutamate release, and decreased glutamate turnover. Our results evidence that an excitotoxic septal lesion leads to increased hippocampal GABA(A) receptors and decreased glutamate neurotransmission. In this situation, a co-ordinated response of hippocampal retaliatory systems takes place to control neuron excitability.

Effects of mercury on neurochemical receptors in wild river otters (Lontra canadensis).

Fish-eating wildlife, such as river otters (Lontra canadensis), accumulate mercury (Hg) at concentrations known to impair animal behavior, but few studies have explored the underlying biochemical changes that precede clinical neurotoxicity. The objective of this study was to determine if Hg exposure can be related to concentrations of neurochemical receptors in river otters. River otter carcasses (n = 66) were collected in Ontario and Nova Scotia (Canada) by local trappers in 2002-2004. Concentrations of Hg (total and organic) were measured in the cerebral cortex and cerebellum. Saturation binding curves for the cholinergic muscarinic acetylcholine (mACh) receptor and dopamine-2 (D2) receptor were completed for each animal to calculate receptor density (Bmax) and ligand affinity (Kd). Negative correlations were found between concentrations of Hg and mACh receptor Bmax (r(total) Hg = -0.458, r(inorganic) Hg = -0.454, r(organic) Hg = -0.443) in the cerebral cortex. A negative correlation was also found between concentrations of total Hg and D2 receptor Bmax (r = -0.292) in the cerebral cortex. These results suggest that neurochemical receptors may prove useful as novel biomarkers of Hg exposure and neurotoxic effects in wildlife. Given the importance of cholinergic and dopaminergic systems in animal physiology, the ecological implications of these changes need to be investigated.

Neuronal nitric oxide synthase activity in rat urinary bladder detrusor: participation in M3 and M4 muscarinic receptor function.

1. The aim of this paper was to determine the different signalling cascades involved in contraction of the rat urinary bladder detrusor muscle mediated via muscarinic acetylcholine receptors (muscarinic AChR). Contractile responses, phosphoinositides (IPs) accumulation, nitric oxide synthase (NOS) activity and cyclic GMP (cGMP) production were measured to determine the reactions associated with the effect of cholinergic agonist carbachol. The specific muscarinic AChR subtype antagonists and different inhibitors of the enzymatic pathways involved in muscarinic receptor-dependent activation of NOS and cGMP were tested. 2. Carbachol stimulation of M(3) and M(4) muscarinic AChR increased contractility, IPs accumulation, NOS activity and cGMP production. All of these effects were selectively blunted by 4-DAMP and tropicamide, M(3) and M(4) antagonists respectively. 3. The inhibitors of phospholipase C (PLC), calcium/calmodulin (CaM), neuronal NOS (nNOS) and soluble guanylate cyclase, but not of protein kinase C and endothelial NOS (eNOS), inhibited the carbachol action on detrusor contractility. These inhibitors also attenuated the muscarinic receptor-dependent increase in cGMP and activation of NOS. 4. In addition, sodium nitroprusside and 8-bromo-cGMP, induced negative relaxant effect. 5. The results obtained suggest that carbachol activation of M(3) and M(4) muscarinic AChRs, exerts a contractile effect on rat detrusor that is accompanied by an increased production of cGMP and nNOS activity. The mechanism appears to occur secondarily to stimulation of IPs turnover via PLC activation. This in turn, triggers cascade reactions involving CaM, leading to activation of nNOS and soluble guanylate cyclase. They, in turn, exert a modulator inhibitory cGMP-mediated mechanism limiting the effect of muscarinic AChR stimulation of the bladder.

An interspecies comparison of mercury inhibition on muscarinic acetylcholine receptor binding in the cerebral cortex and cerebellum.

Mercury (Hg) is a ubiquitous pollutant that can disrupt neurochemical signaling pathways in mammals. It is well documented that inorganic Hg (HgCl(2)) and methyl Hg (MeHg) can inhibit the binding of radioligands to the muscarinic acetylcholine (mACh) receptor in rat brains. However, little is known concerning this relationship in specific anatomical regions of the brain or in other species, including humans. The purpose of this study was to explore the inhibitory effects of HgCl(2) and MeHg on [(3)H]-quinuclidinyl benzilate ([(3)H]-QNB) binding to the mACh receptor in the cerebellum and cerebral cortex regions from human, rat, mouse, mink, and river otter brain tissues. Saturation binding curves were obtained from each sample to calculate receptor density (B(max)) and ligand affinity (K(d)). Subsequently, samples were exposed to HgCl(2) or MeHg to derive IC50 values and inhibition constants (K(i)). Results demonstrate that HgCl(2) is a more potent inhibitor of mACh receptor binding than MeHg, and the receptors in the cerebellum are more sensitive to Hg-mediated mACh receptor inhibition than those in the cerebral cortex. Species sensitivities, irrespective of Hg type and brain region, can be ranked from most to least sensitive: river otter > rat > mink > mouse > humans. In summary, our data demonstrate that Hg can inhibit the binding [(3)H]-QNB to the mACh receptor in a range of mammalian species. This comparative study provides data on interspecies differences and a framework for interpreting results from human, murine, and wildlife studies.

Pharmacological profiles of an anticholinergic agent, phencynonate hydrochloride, and its optical isomers.

AIM: To comparatively study the pharmacological profiles of 3-methyl-3-azabicyclo(3,3,1)nonanyl-9-alpha-yl-alpha-cyclopentyl-alpha-phenyl-alpha-glycolate (phencynonate hydrochloride, CPG), an anticholinergic agent, and its enantiomers [R(-)-and S(+)-CPG]. METHODS: The affinity and relative efficacy were tested using radioligand-binding assay with muscarinic acetylcholine receptors from rat cerebral cortex. The pharmacological activities were assessed in three individual experiments: (1) potentiating the effect of subthreshold hypnotic dose of sodium pentobarbital; (2) inhibiting oxotremorine-induced salivation; and (3) inhibiting the contractile response to carbachol. RESULTS: The order of potency of phencynonate hydrochloride and its optical isomers to inhibit the binding of [3H]quinuclidinyl benzilate ([3H]QNB) was R(-)-CPG (K(i)=46.49+/-1.27 nmol/L)>CPG(K(i)=271.37+/-72.30nmol/L)>S(+)-CPG(K(i)=1263.12+/-131.64 nmol/L). The results showed that R(-)-CPG had the highest affinity to central muscarinic receptors among the three compounds, but did not show any central depressant effects at dose from 10.00 to 29.15 mg/kg. CPG increased the effects of subthreshold hypnotic dose of sodium pentobarbital induced-sleeping [the ED50+/-95% LC value was 21.06+/-3.04 mg/kg]. CPG and R(-)-CPG displayed nearly equipotent effect in depressing oxotremorine-induced salivation [the ED50 +/-95% LC for R(-) and CPG were 1.10+/-0.28 and 1.07+/-0.15 mg/kg, respectively], and the contractile response to carbachol (pA(2) values for R (-) and CPG were 6.84 and 6.80, respectively). S(+)-CPG presented the lowest anticholinergic profiles, but could potentate effects of its enantiomers in some manner. CONCLUSIONS: These data suggested that R(-)-CPG acted as an eutomer in racemate and a competitive antagonist to acetylcholine muscarinic receptors, but S(+)-CPG was less active in comparison to R(-)-CPG and its racemate. The central depressant effects of R(-)-CPG and S(+)-CPG were lower in comparison to its racemate.

Characterization of radioligand binding to a transmembrane receptor reconstituted into Lipobeads.

Lipobeads are hydrogel beads surrounded by a lipid bilayer membrane and have been developed to act as a cell analogue. The FLAG-tagged M(2) muscarinic receptor was incorporated onto the surface of the Lipobead by incubating pre-Lipobeads with proteoliposomes containing the receptor. Receptors reconstituted onto the surface of the Lipobeads were functional in that they bound the antagonists quinuclidinylbenzilate and scopolamine with characteristic muscarinic affinities. This demonstrates the feasibility of using Lipobeads to study the binding properties of the M(2) muscarinic receptor and offers a promising approach to the study of transmembrane protein biology in general.

[Screening method for compounds acting at super low concentrations]. / Metod skrininga veshchestv, deistvuiushchikh v sverkhmalykh kontsentratsiiakh.

At present, there are no simple and reliable methods for selection of compounds with physiological activity in super-low concentrations (SLC). The screening system with high predictability is proposed. At the beginning, experiment is carried out on infusorians--spirostoma. Their motion activity, behavior, living ability and shape are registered at concentrations of substances in a range of 10(-3)-10(-13) mol/l. If the effect is detected on spirostoma, then experiments are performed on laboratory animals or their isolated tissues. Specific deviations on injection substance in SLC are detected, depending on compounds class. Nine representatives of different classes of biologically active compounds are investigated in the work: carbofos, deltametrin, 3-quinuclidinyl benzylate, atropine, phenosan, fenazepam, dopamine receptor antagonist SCH-23390, ciprine, and hexenal. It is accepted that if compound action in SLC is observed on spirostoma, then effects in super-low doses are also detected in a whole animal.